PPK: Radiation Response and Cell Proliferation

癌症的一个特点是失去了控制细胞增殖的调节机制。在减弱或丧失的控制中，有一种能力是阻止细胞周期进程，以应对电离辐射和随之而来的DNA损伤或对细胞的其他侮辱。这些检查点包括G1/S交界处、S期、G2/M过渡期和有丝分裂纺锤体集合点的停滞。Polo激酶是一类在蛋白质的羧基末端保留进化保守的氨基酸序列的蛋白激酶家族，称为Polo box。它们参与了细胞从G2晚期进入有丝分裂的过程，通过有丝分裂和退出有丝分裂。在本申请中检测的Polo激酶是Plk3(以前由PRK指定)，它是已知的三种哺乳动物Polo激酶之一，其功能尚不清楚。我们已经证明了这种蛋白在整个细胞周期中相对持续地表达，但发现它的激酶活性是波动的。基于免疫共沉淀研究，它与同相促进复合体(APC)有关，但与其相互作用的蛋白质尚不清楚。对电离辐射和DNA损伤的反应也非常迅速，提示它在细胞周期停滞或DNA修复中发挥作用，在细胞暴露于诺可达唑后，它变得磷酸化，提示在纺锤体组装检查点中发挥作用。奇怪的是，与其近亲Plk1不同，Plk1在转染后转化NIH 3T3细胞，Plk3导致哺乳动物细胞在转染后发生凋亡，并在胞质分裂期间阻止细胞。虽然Plk3的过度表达与有丝分裂后的中体有关，但内源性的Plk3并非如此，这表明对Plk3过度表达的结果的解释可能不能准确地反映Plk3的真实功能(S)。这项应用的直接目标是确定Plk3的特征，并阐明其在细胞调节以及应对辐射损伤和其他侮辱中的功能(S)。具体地说，我们建议建立辐射和诺康唑处理后Plk3的磷酸化模式，确定其激酶活性的底物和与其相互作用的其他蛋白质，并分离能够绕过细胞凋亡并允许在转染后存活的突变体Plk3。利用这些信息和Plk3基因突变或缺失的小鼠胚胎成纤维细胞，我们建议阐明Plk3参与细胞增殖调节以及对辐射和随后的DNA损伤的反应的途径(S)。

One hallmark of cancer is the loss of regulatory mechanisms that control cell proliferation. Among the controls that are attenuated or lost is the capacity to arrest cell cycle progression in response to ionizing radiation and consequent DNA damage or other insult to the cell. Such checkpoints include arrest at the G1/S boundary, during the S phase, during the G2/M transition, and at the point of mitotic spindle assembly. The Polo kinases are a family of kinases that retain an evolutionarily conserved amino acid sequence, designated the polo box, at the carboxy end of the protein. They participate in the transit of cells from late G2 into mitosis, through mitosis and exit from mitosis. The polo kinase examined in this application is Plk3 (former by designated Prk), one of three known mammalian polo kinases, whose function remains ill defined. We have shown that this protein is expressed relatively constantly throughout the cell cycle but find that its kinase activity fluctuates. Based on co-immunoprecipitation studies, it associates with the amphase promoting complex (APC), but the proteins with which it interacts are not known. It is also very rapidly phosphorylated in response to ionizing radiation and DNA damage, suggestive of a role in cell cycle arrest or DNA repair, and it becomes phosphorylated following exposure of cells to nocodazole, suggestive of a role in the spindle assembly checkpoint. Curiously, unlike its close relative Plk1, which transforms NIH 3T3 cells following transfection, Plk3 causes mammalian cells to undergo apoptosis following transfection and arrests cells during cytokinesis. Whereas overexpressed Plk3 associates with a post-mitotic midbody, endogenous Plk3 does not, suggesting that interpretation of results derived from Plk3 overexpression may not accurately reflect the Plk3 true function(s). The immediate goals of this application are to characterize Plk3 and to elucidate its function(s) in cellular regulation and in response radiation damage and other insult. Specifically we propose to establish phosphorylation patterns of Plk3 following irradiation and nocodazole treatment, identify substrates for its kinase activity and other proteins with which it interacts, and isolate mutant Plk3 that circumvent apoptosis and permit survival following transfection. Using this information and mouse embryo fibroblasts with mutant or null Plk3 alleles, we propose to elucidate the pathway(s) in which Plk3 participates in the regulation of cell proliferation and in response to radiation and consequent DNA damage.